Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

Abstract

Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (∼10 μM). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins.G ene expression control occurs through the combined activities of transcription factors and chromatin regulators, considered as effectors of epigenetic mechanisms. Posttranslational modifications of nucleosomal histones, DNA methylation, local compaction, and long-range interactions determine a variety of chromatin structures that can affect the transcriptional output.A group of chromatin regulators are the Polycomb Repressive complexes (PRCs) (1, 2). These Polycomb group (PcG) proteins are encoded by genes initially discovered over 60 years ago as repressors of the Hox genes in Drosophila (3). The PcG proteins form two main silencing heteromeric complexes called PRC1 and PRC2; both are highly conserved in eukaryotes and either in isolation or synergistically can silence genes (4, 5). The catalytic functions of both complexes include ubiquitin-ligase (H3K119ub1) and methyltransferase activity (H3K27Me3), respectively. In mammals, the PRC2 core is formed by, at least, four heteromeric units, one of which is EZH2 (or its paralog, EZH1), the methyltransferase that catalyzes the trimethylation of histone H3 at Lys27 (5). This modification can act as an anchoring site of PRC1 complexes containing chromobox (CBX) proteins. These CBX proteins recruit PRC1 complex onto PRC2-enriched chromatin, facilitating the monoubiquitylation. The PRC1-dependent monoubiquitylation at Lys119 of histone H2A correlates with transc...

Ligand-based design identifies a potent NUPR1 inhibitor exerting anticancer activity via necroptosis

“…Fragments of the simulated protein structure with a length of 5 or 7 amino acid residues were used as receptors for molecular docking of either TFP or TFP-derived compounds, by using the AutoDock Vina software (45). The choice of using short fragments of NUPR1 was motivated not only by the necessity of reducing its exceedingly large conformational space, but also by the fact that ligand binding to the protein is dictated by local hydrophobicity spanning up to 7 amino acid residues (27,29). We previously proved that molecular docking is sufficiently accurate to reveal the binding locations of drugs to NUPR1 in a blind search (29).…”

“…Structurally, NUPR1 is an intrinsically disordered protein (IDP) with an entirely disordered conformation (5,(25)(26)(27)(28). Consequently, the target-based high throughput screening for drug selection toward this protein is highly challenging.…”

Amphipathic helical peptides hamper protein-protein interactions of the intrinsically disordered chromatin nuclear protein 1 (NUPR1)

“…It plays key roles in pancreatic tumorigenesis, acting downstream of the Kras G12D oncogene mutation, which is critical for pancreatic carcinogenesis (19). Furthermore, NUPR1 is involved in apoptosis by forming a complex with prothymosin  (20), another IDP, as well as implicated in DNA binding (21) and repair (22), through interaction with the male specific lethal protein (MSL1), and in the association to proteins of the Polycomb group (23). All these interactions provide the rationale to consider NUPR1 inhibition as a clinically feasible strategy to expand the PDAC drug arsenal.…”

“…We have shown that binding to all those biomolecules occurs through two distant regions of the NUPR1 sequence (20,(22)(23)(24): (i) the hydrophobic patch around Ala33, containing the two aromatic rings of the protein, Tyr30 and Tyr36; and, (ii) the region around Thr68, which is a polypeptide patch with relatively high hydrophobicity. The Ala33Gln and Thr68Gln mutants have both shown impaired binding to NUPR1 protein partners, therefore pinpointing the importance of those regions (23). As tested in PDAC-derived cell-based assays (24), both regions also intervene in the binding of small molecules capable of inducing cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, thus mimicking NUPR1-deficiency.…”

Targeting HIF-1α Function in Cancer through the Chaperone Action of NQO1

“…A cell-based assay would also be required to test hits from the screen under in vivo conditions, for example proteins labeled with FRET donors and acceptors, co-immunoprecipitation or indirectly by measuring amounts of HIF1α. Although challenging due to several highly disordered regions in the HIF-1 protein, such screening would be still feasible as recently reported for inhibiting PPI in the cancer-associated and intrinsically disordered protein NUPR1 [148,149].…”